Search

Abstract Background Abdominal aortic aneurysm (AAA) is a complex disorder with multiple genetic risk factors. Using affected relative pair linkage analysis, we previously identified an AAA susceptibility locus on chromosome 19q13. This locus has been designated as the AAA1 susceptibility locus in the Online Mendelian Inheritance in Man (OMIM) database. Methods Nine candidate genes were selected from the AAA1 locus based on their function, as well as mRNA expression levels in the aorta. A sample of 394 cases and 419 controls was genotyped for 41 SNPs located in or around the selected nine candidate genes using the Illumina GoldenGate platform. Single marker and haplotype analyses were performed. Three genes (CEBPG, PEPD and CD22) were selected for DNA sequencing based on the association study results, and exonic regions were analyzed. Immunohistochemical staining of aortic tissue sections from AAA and control individuals was carried out for the CD22 and PEPD proteins with specific antibodies. Results Several SNPs were nominally associated with AAA (p < 0.05). The SNPs with most significant p-values were located near the CCAAT enhancer binding protein (CEBPG), peptidase D (PEPD), and CD22. Haplotype analysis found a nominally associated 5-SNP haplotype in the CEBPG/PEPD locus, as well as a nominally associated 2-SNP haplotype in the CD22 locus. DNA sequencing of the coding regions revealed no variation in CEBPG. Seven sequence variants were identified in PEPD, including three not present in the NCBI SNP (dbSNP) database. Sequencing of all 14 exons of CD22 identified 20 sequence variants, five of which were in the coding region and six were in the 3'-untranslated region. Five variants were not present in dbSNP. Immunohistochemical staining for CD22 revealed protein expression in lymphocytes present in the aneurysmal aortic wall only and no detectable expression in control aorta. PEPD protein was expressed in fibroblasts and myofibroblasts in the media-adventitia border in both aneurysmal and non-aneurysmal tissue samples. Conclusions Association testing of the functional positional candidate genes on the AAA1 locus on chromosome 19q13 demonstrated nominal association in three genes. PEPD and CD22 were considered the most promising candidate genes for altering AAA risk, based on gene function, association evidence, gene expression, and protein expression.

Abstract Background Abdominal aortic aneurysms are a common disorder with an incompletely understood etiology. We used Illumina and Affymetrix microarray platforms to generate global gene expression profiles for both aneurysmal (AAA) and non-aneurysmal abdominal aorta, and identified genes that were significantly differentially expressed between cases and controls. Results Affymetrix and Illumina arrays included 18,057 genes in common; 11,542 (64%) of these genes were considered to be expressed in either aneurysmal or normal abdominal aorta. There were 3,274 differentially expressed genes with a false discovery rate (FDR) ≤ 0.05. Many of these genes were not previously known to be involved in AAA, including SOST and RUNX3, which were confirmed using Q-RT-PCR (Pearson correlation coefficient for microarray and Q-RT-PCR data = 0.89; p-values for differences in expression between AAA and controls for SOST: 4.87 × 10-4 and for RUNX3: 4.33 × 10-5). Analysis of biological pathways, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG), indicated extreme overrepresentation of immune related categories. The enriched categories included the GO category Immune Response (GO:0006955; FDR = 2.1 × 10-14), and the KEGG pathways natural killer cell mediated cytotoxicity (hsa04650; FDR = 5.9 × 10-6) and leukocyte transendothelial migration (hsa04670; FDR = 1.1 × 10-5). Conclusion Previous studies have provided evidence for the involvement of the immune system in AAA. The current expression analysis extends these findings by demonstrating broad coordinate gene expression in immunological pathways. A large number of genes involved in immune function were differentially expressed in AAA, and the pathway analysis gave these results a biological context. The data provide valuable insight for future studies to dissect the pathogenesis of human AAA. These pathways might also be used as targets for the development of therapeutic agents for AAA.

Charcot-Marie-Tooth Disease, Subtype 4J (CMT4J), and the severe multisystem Yunis-Varon Syndrome (YVS) are caused by biallelic loss-of-function variants in FIG4 , the gene encoding polyphosphoinositide phosphatase. Patients carrying one allele with residual function typically manifest a milder peripheral sensory and motor neuropathy (CMT4J). In contrast, complete loss of function in both alleles leads to YVS, characterized by dysmorphism and profound neurological, skeletal, and cardiovascular abnormalities, often resulting in early death. With the increased accessibility of genetic testing, recent case reports document a broadening range of clinical features encompassing neuropathy, leukodystrophy, brain malformations, skeletal abnormalities, respiratory impairment and Parkinsonism in patients with disease causing FIG4 variants. Here, we analyze the literature with a focus on clinically and genetically characterized cases with FIG4 loss of function. Among a total of 67 identified patients, 60 (90%) were classified as CMT4J and 7 (10%) as YVS. Neuropathy was documented in 60 cases (90%), neurodevelopmental disability in 18 (27%) parkinsonism 8 (12%), scoliosis 9 (13%), and other bone malformations 17 (25%). Of 32 patients with MRI imaging of the brain, 10/32 (31%) displayed atrophic changes or volume loss and 11/32 (34%) had white matter changes. We propose that loss-of-function variants in FIG4 produce a spectrum of disease anchored at each end by CMT4J and YVS, between which a defined range of phenotypes exists. As FIG4 gene replacement therapy shows great promise in preclinical studies and is expected to transition into human studies, there is an urgent need for a comprehensive phenotypic characterization of FIG4 loss of function related diseases to facilitate clinical trial readiness through tailored prospective natural history data collection. [ABSTRACT FROM AUTHOR]

Fuel surrogates are mixtures that mimic the properties of real fuels with only a small number of components, simplifying the calculation and simulation of fuel-related processes. This work extends a previously published surrogate optimization algorithm toward the generation of fuel surrogates with a focus on liquid-liquid extraction characteristics. For this purpose, experimental liquid-liquid equilibrium data from batch extraction experiments are incorporated into the calculation procedure as an additional constraint. The use of the method is demonstrated by optimizing a surrogate for the catalytic reformate. Application of the surrogate to an extraction process and comparison with experimental data demonstrate that the resulting surrogate accurately depicts the properties of the real mixture with regard to liquid-liquid extraction performance. This demonstrates that the use of such surrogates is of particular interest for mixtures used as extracting agents for biofuels., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Obtaining plasma from a blood sample and preparing it for subsequent analysis is currently a laborious process involving experienced health-care professionals and centrifugation. We circumvent this by utilizing capillary forces and microfluidic engineering to develop an autonomous plasma sampling device that filters and stores an exact amount of plasma as a dried plasma spot (DPS) from a whole blood sample in less than 6 min. We tested 24 prototype devices with whole blood from 10 volunteers, various input volumes (40-80 μL), and different hematocrit levels (39-45%). The resulting mean plasma volume, assessed gravimetrically, was 11.6 μL with a relative standard deviation similar to manual pipetting (3.0% vs 1.4%). LC-MS/MS analysis of caffeine concentrations in the generated DPS (12 duplicates) showed a strong correlation ( R 2 = 0.99) to, but no equivalence with, concentrations prepared from corresponding plasma obtained by centrifugation. The presented autonomous DPS device may enable patient-centric plasma sampling through minimally invasive finger-pricking and allow generatation of volume-defined DPS for quantitative blood analysis.

Dried blood spot (DBS) sampling is a promising method for collection of microliter blood samples. However, hematocrit-related bias in combination with subpunch analysis can result in inaccurate quantification of analytes in DBS samples. In this study we use a microfluidic DBS card, designed to automatically collect fixed volume DBS samples irrespective of the blood hematocrit, to measure caffeine concentration in normal finger prick samples obtained from 44 human individuals. Caffeine levels originating from blood drops of unknown volume collected on the volumetric microfluidic DBS card were compared to volume-controlled pipetted DBS samples from the same finger prick. Hematocrit independence and volumetric sampling performances were also verified on caffeine-spiked blood samples in vitro, using both LC-MS/MS and gravimetric methods, on hematocrits from 26 to 62%. The gravimetric measurements show an excellent metering performance of the microfluidic DBS card, with a mean blood sample volume of 14.25 μL ± 3.0% ( n = 51). A measured mean bias below 2.9% compared to normal hematocrit (47%) demonstrates that there is no significant hematocrit-induced bias. LC-MS/MS measurements confirm low CV and hematocrit independence of the sampling system and exhibit no substantial mean bias compared to pipetted DBS. Tests with 44 individuals demonstrated applicability of the microfluidic DBS card for direct finger prick blood sampling, and measured caffeine concentrations show a good agreement with measurements of pipetted DBS. The presented concept demonstrates a good volumetric performance which can help to improve the accuracy of DBS analysis by analyzing a whole spot, equivalent to a defined volume of liquid blood.

Whole-blood microsampling provides many benefits such as remote, patient-centric, and minimally invasive sampling. However, blood plasma, and not whole blood, is the prevailing matrix in clinical laboratory investigations. The challenge with plasma microsampling is to extract plasma volumes large enough to reliably detect low-concentration analytes from a small finger prick sample. Here we introduce a passive plasma filtration device that provides a high extraction yield of 65%, filtering 18 μL of plasma from 50 μL of undiluted human whole blood (hematocrit 45%) within less than 10 min. The enabling design element is a wedge-shaped connection between the blood filter and the hydrophilic bottom surface of a capillary channel. Using finger prick and venous blood samples from more than 10 healthy volunteers, we examined the filtration kinetics of the device over a hematocrit range of 35-55% and showed that 73 ± 8% of the total protein content was successfully recovered after filtration. The presented plasma filtration device tackles a major challenge toward patient-centric blood microsampling by providing high-yield plasma filtration, potentially allowing reliable detection of low-concentration analytes from a blood microsample.

Lysosomes receive and degrade cargo from endocytosis, phagocytosis and autophagy. They also play an important role in sensing and instructing cells on their metabolic state. The lipid kinase PIKfyve generates phosphatidylinositol-3,5-bisphosphate to modulate lysosome function. PIKfyve inhibition leads to impaired degradative capacity, ion dysregulation, abated autophagic flux and a massive enlargement of lysosomes. Collectively, this leads to various physiological defects, including embryonic lethality, neurodegeneration and overt inflammation. The reasons for such drastic lysosome enlargement remain unclear. Here, we examined whether biosynthesis and/or fusion-fission dynamics contribute to swelling. First, we show that PIKfyve inhibition activates TFEB, TFE3 and MITF, enhancing lysosome gene expression. However, this did not augment lysosomal protein levels during acute PIKfyve inhibition, and deletion of TFEB and/or related proteins did not impair lysosome swelling. Instead, PIKfyve inhibition led to fewer but enlarged lysosomes, suggesting that an imbalance favouring lysosome fusion over fission causes lysosome enlargement. Indeed, conditions that abated fusion curtailed lysosome swelling in PIKfyve-inhibited cells., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Phosphatidylethanol (PEth) is a group of phospholipids formed in cell membranes following alcohol consumption. PEth measurement in whole blood samples is established as a specific alcohol biomarker with clinical and medico-legal applications. This study further evaluated the usefulness of dried blood spot (DBS) samples collected on filter paper for PEth measurement. Specimens used were surplus volumes of venous whole blood sent for routine LC-MS/MS quantification of PEth 16:0/18:1, the major PEth homolog. DBS samples were prepared by pipetting blood on Whatman 903 Protein Saver Cards and onto a volumetric DBS device (Capitainer). The imprecision (CV) of the DBS sample amount based on area and weight measurements of spot punches were 23-28%. Investigation of the relationship between blood hematocrit and PEth concentration yielded a linear, positive correlation, and at around 1.0-1.5μmol/L PEth 16:0/18:1, the PEth concentration increased by ~0.1μmol/L for every 5% increase in hematocrit. There was a close agreement between the PEth concentrations obtained with whole blood samples and the corresponding results using Whatman 903 (PEth DBS =1.026 PEth WB +0.013) and volumetric device (PEth DBS =1.045 PEth WB +0.016) DBS samples. The CV of PEth quantification in DBS samples at concentrations≥0.05μmol/L were ≤15%. The present results further confirmed the usefulness of DBS samples for PEth measurement., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Inhomogeneous sample distribution in DBS is a problem for accurate quantitative analysis of DBS, and has often been explained by chromatographic effects., Results: We present a model describing formation of inhomogeneous DBS during drying of the spot caused by higher evaporation rates of water at the edge as compared with the center. Color intensity analysis shows that the relative humidity and DBS card position affect the homogeneity of DBS., Conclusion: The so-called 'coffee-stain effect' explains the typical distribution pattern of analytes with higher concentrations measured along the edge of DBS as compared with the center. The driving mechanism and potential influencing factors should be considered when addressing the inhomogeneity of DBS in the future.

Background: DBS samples collected from a fingerprick typically vary in volume and homogeneity and hence make an accurate quantitative analysis of DBS samples difficult., Results: We report a prototype which first defines a precise liquid volume and subsequently stores it to a conventional DBS matrix. Liquid volumes of 2.2 µl ± 7.1% (n = 21) for deionized water and 6.1 µl ± 8.8% (n = 15) for whole blood have been successfully metered and stored in DBS paper., Conclusion: The new method of collecting a defined volume of blood by DBS sampling has the potential to reduce assay bias for the quantitative evaluation of DBS samples while maintaining the simplicity of conventional DBS sampling.

Lycopersicon esculentum seeds germinate after rehydration in complete darkness. This response was inhibited by a far-red light (FR) pulse, and the inhibition was reversed by a red light (R) pulse. Comparison of germination in phytochrome-deficient mutants (phyA, phyB1, phyB2, phyAB1, phyB1B2 and phyAB1B2) showed that phytochrome B2 (PhyB2) mediates both responses. The germination was inhibited by strong continuous R (38 micromol m(-2) s(-1)), whereas weak R (28 nmol m(-2) s(-1)) stimulated seed germination. Hourly applied R pulses of the same photon fluence partially replaced the effect of strong continuous R. This response was called 'antagonistic' because it counteracts the low fluence response (LFR) induced by a single R pulse. This antagonistic response might be an adaptation to a situation where the seeds sit on the soil surface in full sunlight (adverse for germination), while weak R might reflect that situation under a layer of soil. Unexpectedly, the effects of continuous R or repeated R pulses were mediated by phytochrome A (PhyA). We therefore suggest that low levels of PhyA in its FR-absorbing form (Pfr) cause inhibition of seed germination produced either by extended R irradiation (by degradation of PhyA-Pfr) or by extended FR irradiation [keeping a low Pfr/R-absorbing form (Pr) ratio].

Unlabelled: Aim of the study was to evaluate the influence of different spiral-CT parameters for the visualisation of vascular stenoses, especially of the renal arteries., Material and Methods: Models with a density equivalent to that of fat, filled with diluted contrast agent, and an inner lumen of 4, 6, 8 mm were scanned in x-, y- and z-direction. Data were acquired in up to 24 second long spiral-CT scans using different spiral-CT parameters (collimation, table speed, reconstruction algorithm, tube current). Detection of the degree of stenosis was achieved by assessment of the axial images and 3D reconstructions., Results: The best correlation between real and measured degree of stenosis was seen by using a small collimation, a low table increment and assessment of the axial images reconstructed in standard algorithm. The stenosis degrees of models directed in x- and y-direction were overestimated and those in z-direction were underestimated depending on the spiral-CT parameters., Conclusion: For optimal imaging of renal artery stenoses, collimation of 2 mm (pitch = 1-2 and a reconstruction interval of 1 mm is recommended.